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Biomedical subjects

S M Wu

Publications and source records attributed to S M Wu.

At least 73 records · Page 4Linked to original sources
PubMed1998

Characterization of spontaneous inhibitory synaptic currents in salamander retinal ganglion cells.

Spontaneous and light-evoked postsynaptic currents (sPSCs and lePSCs, respectively) in retinal ganglion cells of the larval tiger salamander were recorded under voltage-clamp conditions from living retinal slices. The focus of this study is to characterize the spontaneous inhibitory PSCs (sIPSCs) and their contribution to the light-evoked inhibitory PSCs (leIPSCs) in ON-OFF ganglion cells. sIPSCs were isolated from spontaneous excitatory PSCs (sEPSCs) by application of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX) + 50 microM 2-amino-5-phosphonopentanoic acid (AP5). In approximately 70% of ON-OFF ganglion cells, bicuculline (or picrotoxin) completely blocks sIPSCs, suggesting all sIPSCs in these cells are mediated by GABAergic synaptic vesicles and gamma-aminobutyric acid-A (GABAA) receptors (GABAergic sIPSCs, or GABAsIPSCs). In the remaining 30% of - ganglion cells, bicuculline (or picrotoxin) blocks 70-98% of the sIPSCs, and the remaining 2-30% are blocked by strychnine (glycinergic sIPSCs, or GLYsIPSCs). GABAsIPSCs occur randomly with an exponentially distributed interval probability density function, and they persist without noticeable rundown over time. The GABAsIPSC frequency is greatly reduced by cobalt, consistent with the idea that they are largely mediated by calcium-dependent vesicular release. GABAsIPSCs in DNQX + AP5 are tetrodotoxin (TTX) insensitive, suggesting that amacrine cells that release GABA under these conditions do not generate spontaneous action potentials. The average GABAsIPSCs exhibited linear current-voltage relation with a reversal potential near the chloride equilibrium potential, and an average peak conductance of 319.67 +/- 252.83 (SD) pS. For GLYsIPSCs, the average peak conductance increase is 301.68 +/- 94.34 pS. These parameters are of the same order of magnitude as those measured in inhibitory miniature postsynaptic currents (mIPSCs) associated with single synaptic vesicles in the CNS. The amplitude histograms of GABAsIPSCs did not exhibit multiple peaks, suggesting that the larger events are not discrete multiples of elementary events (or quanta). We propose that each GABAsIPSC or GLYsIPSC in retinal ganglion cells is mediated by a single or synchronized multiple of synaptic vesicles with variable neurotransmitter contents. In a sample of 16 ON-OFF ganglion cells, the average peak leIPSC (held at 0 mV) at the light onset is 509.0 +/- 233.85 pA and that at the light offset is 529.0 +/- 339.88 pA. The approximate number of GABAsIPSCs and GLYsIPSCs required to generate the average light responses, calculated by the ratio of the charge (area under current traces) of the leIPSCs to that of the average single sIPSCs, is 118 +/- 52 for the light onset, and 132 +/- 76 for the light offset.

Ambystoma↗
PubMed1998

The effectiveness and factors related to treatment results of gamma knife radiosurgery for meningiomas.

A retrospective analysis was conducted on 80 patients with intracranial mengiomas treated with Gamma Knife radiosurgery between 1993 and 1996. The purpose was to analyze the efficacy of the treatment and to assess appropriate treatment parameters. The results were assessed by regular MR examinations, and tumor volume was measured at 6-month intervals. Mean follow-up duration was 21 months (range 6-45 months). 63 meningiomas were at the skull base and 17 were distal from the skull base. Tumor volumes <5 ml (n=38), 5-10 ml (n=21), 10-15 ml (n=14), 15-20 ml (n=7). The patients were divided into 3 groups according to the radiation dose. The groups were high-dose (peripheral dose 17-20 Gy, n=19), medium-dose (15-16 Gy, n=33) and low-dose (12-14 Gy, n=28) groups. A statistical method (Generalized Estimation Equation) was applied to compare treatment results in these groups with different doses and tumor volumes. The volume measurement at the latest follow-up showed 74% (59/80) meningiomas decreased in volume, 17% (14/80) had no tumor enlargement and 9% (7/80) had increased in volume. The increased volume was found more frequently in the patients with a short (6-12 months) follow-up period. In this series, the tumors had 32&percnt reduction in average tumor volume at 3 years after radiosurgery. At the range of 12-20 Gy peripheral dose (PD), radiosurgery was effective to reduce tumor volume 0.7% per month (p<0.05). However, higher doses had no significant difference on tumor volume reduction (p>0.05). On the other hand, high-dose (PD>17 Gy) treatment was associated with a higher risk of temporary tumor swelling and the development of adverse radiation effects (AREs). The AREs detected on MR images occurred in (25/80) 31% patients. Only 6/25 AREs were symptomatic and 2 had neurological sequelae. Peripheral doses, tumor volumes and their locations had significant impacts on the ARE (p<0. 05). In conclusion, a peripheral dose of 15-16 Gy may be adequate for meningiomas with small volumes (<5 ml). In larger tumors (>10 ml) a lower PD is preferred (12-14 Gy). To avoid initial tumor swelling and ARE, high-dose irradiation (PD>17 Gy) is not recommended for meningiomas larger than 5 ml.

Adult↗
PubMed1998

Inactivation of the luteinizing hormone/chorionic gonadotropin receptor by an insertional mutation in Leydig cell hypoplasia.

We previously identified a nonsense mutation (Cys545Stop) in the paternal human LH/CG receptor (hLHR) allele in a family with two 46,XY children afflicted with Leydig cell hypoplasia. This mutation abolished the signal transduction capability of the affected hLHR. We have now examined all coding exons and the transcript of both alleles of the hLHR gene of the affected children. A 33-bp in-frame insertion was found in the maternal hLHR allele. This insertion occurred between nucleotide 54 and 55 and might be the result of a partial gene duplication. Genomic DNA-PCR showed that this defective maternal hLHR allele was inherited by the two affected children. However, examination of the inheritance of the 935-A/G polymorphism of the hLHR by genomic- and RT-PCR indicated that the maternal hLHR allele was not expressed in cultured fibroblasts of the patients. The effect of the in-frame insertion on the biological activity of the hLHR was examined by expressing the mutated hLHR construct, generated by site-directed mutagenesis, in HEK 293 cells. The expression of the mRNA for the mutant hLHR in HEK 293 cells was not affected. Response of cells expressing the mutated hLHR to hCG stimulation was impaired as demonstrated by reduced intracellular cAMP biosynthesis. This change in signal transduction was the result of a profound reduction in hormone binding at the cell surface due to altered expression and processing of the mutated receptor. We conclude that Leydig cell hypoplasia in this family is the result of compound heterozygous loss-of-function mutations of the hLHR gene.

Alleles↗
PubMed1998

Testicular seminoma in a patient with a constitutively activating mutation of the luteinizing hormone/chorionic gonadotropin receptor.

A white man who had been diagnosed, 35 years previously at the age of 27 months, to have precocious puberty, was later determined to have familial male-limited precocious puberty (FMPP), on the basis of his family history, increased serum testosterone, prepubertal concentrations of follicle stimulating hormone and luteinizing hormone, and Leydig cell hyperplasia. Recently, this diagnosis was confirmed by molecular genetic analysis that demonstrated the presence of a heterozygous constitutive activating mutation of the luteinizing hormone/chorionic gonadotropin receptor. This dominant gain-of-function Asp578Gly mutation has been shown constitutively to activate the receptor in the absence of the agonist, leading to enhanced synthesis of cAMP and, in turn, to increased, sustained production of testosterone. In 1994, this patient was found to have a testicular seminoma. He represents the first case of a testicular germ cell tumor described in an FMPP patient, raising the possibility of a potentially harmful effect of prolonged increased concentrations of sex hormones, with onset early in life, upon the cellular components of the testes.

Adult↗
PubMed1997

The impact of day care on ventilation tube insertion.

OBJECTIVE: To study the effect of day care and tube type, as well as other risk factors related to ventilation tube insertion and reinsertion. DESIGN: A case series of 456 consecutive cases with 162 controls from a well-baby examination group evaluated for age, sex, smoking history and day care attendance. SETTING: Private practice in a mid-sized, southeastern university town. PATIENTS AND CONTROLS: This was a referred sample of patients who were entered consecutively in a private-practice setting. All children were age five or less at entry into the study. INTERVENTIONS: Children underwent ventilation tube insertion with or without adenoidectomy, using standing indications. MAIN OUTCOME MEASURES: The primary outcome measures were whether or not a child had the need for a second set of tubes, and also determining the status of the child's ears for at least 1 year after tubes had extruded. RESULTS: Day care and younger age were both identified as risk factors associated with initial ventilation tube insertion. Only 11% (seven out of 63) of home care children, as compared with 31% (108 out of 346) day care children, had the insertion of ventilation tubes (P = 0.000). Day care children who had tubes inserted and adenoidectomy (with or without tonsillectomy) had a significantly lower rate of reintubation than children who had tube insertion alone (P = 0.00). Day care and young age are significant risk factors for any child, both with a first set of tubes and for ventilation tube reinsertion. Children in day care had a reintubation rate of 36% as compared to 11% for those in home care. Parents should be aware that day care can represent a two-fold hazard both in the observed connection between day care and tube insertion and the demonstrated increased probability of reinsertion. Any studies looking at ventilation tube outcomes need to make certain to monitor for day care attendance.

Adenoidectomy↗
PubMed1997

The binding of receptor-recognized alpha2-macroglobulin to the low density lipoprotein receptor-related protein and the alpha2M signaling receptor is decoupled by oxidation.

Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.

Animals↗
PubMed1997

Genomic sequence and transcription start site for the human gamma-glutamyl carboxylase.

The human gene for gamma-glutamyl carboxylase is 13 kb in length and contains 15 exons. Transcription starts at a cytosine 217 base pair upstream of the first codon. There are two major transcripts in all tissues examined. They are distinguished by the presence of an Alu sequence in the 3' nontranslated end of the longer species. Relative mRNA levels for 12 bovine tissues are presented.

Animals↗
PubMed1997

The propeptide binding site of the bovine gamma-glutamyl carboxylase.

gamma-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the gamma-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the gamma-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the gamma-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine gamma-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1997

Interaction between obesity and genetic polymorphisms in the apolipoprotein CIII gene and lipoprotein lipase gene on the risk of hypertriglyceridemia in Chinese.

To understand the effects of the interaction between genetic polymorphisms and obesity on the risk of hypertriglyceridemia (HTG), two polymorphisms, an SstI polymorphism on the apolipoprotein CIII gene and a HindIII polymorphism on the lipoprotein lipase gene, were analyzed in 339 Chinese subjects with (82 cases in the HTG group) or without HTG (257 cases in the control group). Our data revealed that the frequencies of obesity, the SstI minor allele (S2), and the HindIII major allele (H+) in the HTG group were significantly higher than in the control group. Subgroup analysis revealed that the association between these two polymorphisms and HTG occurred predominantly in nonobese subjects and in subjects with the less hypertriglyceridemic genotype of another polymorphism. Multivariate logistic regression analysis showed that all three risk factors (obesity, S2-containing chromosome, and H+ homozygosity) were associated with HTG, and an interaction was found between obesity and H+ homozygosity for the occurrence of HTG. The risk of HTG increased significantly with combinations of risk factors. Subjects can be divided into low or high risk groups for HTG using such combinations. These results provide evidence of interaction between obesity and the HindIII polymorphism of the lipoprotein lipase gene on the risk of HTG.

Alleles↗
PubMed1997

Characterization of glutamate transporter function in the tiger salamander retina.

Glutamate transporters in the tiger salamander retina were studied by autoradiographic and intracellular recording techniques. When the retina was incubated with 15 microM L-[3H]glutamate, photoreceptors and Muller cells were labeled, indicating that these cells had high-affinity glutamate uptake transporters. A much higher dose of glutamate than kainate was required in the bath to produce the same membrane depolarization in horizontal cells (HCs), and the time course of glutamate-induced depolarization was much slower than that of the kainate-induced depolarization. Since glutamate is a substrate of glutamate transporters whereas kainate is not, we attribute these differences to the buffering of extracellular glutamate by glutamate transporters in the retina. D-aspartate (D-asp) increased the efficacy of bath-applied glutamate. Dihydrokainate (DHKA) exerted little effect on glutamate efficacy when applied alone, but it increased glutamate efficacy in the presence of D-asp. These results are consistent with the notion that glutamate transporters in Muller cells are D-asp sensitive and those in photoreceptors are DHKA and D-asp sensitive. Application of DHKA (1-2 mM) did not affect the dark membrane potential or the light responses in rods and cones, but it depolarized the HC dark membrane potential and reduced the HC peak and tail light responses. Our results suggest that DHKA-sensitive glutamate transporters in photoreceptors regulate glutamate levels in rod and cone synaptic clefts. They modulate dark membrane potential and the relative rod cone inputs in retinal HCs.

ATP-Binding Cassette Transporters↗
PubMed1997

Characterization and cellular localization of PSG in rat testis.

In order to establish the rat testis as a model system for studying the human pregnancy-specific beta1-glycoprotein (PSG), expression and cellular distribution of PSG in rat testis were examined. Three partial PSG cDNAs, namely, rnCGM6, rnGCM7, and rnCGM8 were obtained when rat testis cDNA libraries were screened with a human placental PSG cDNA probe. Unlike the human PSGs, the rat PSGs show less nucleotide and amino acid sequence homology among family members. The rat PSGs also have multiple truncated leader sequences followed by immunoglobulin variable-like N domains while human PSGs have a single N domain. Examination of the testis, intestine, kidney, liver, lung, and muscle of male rats by reverse transcription-polymerase chain reaction (RT-PCR) with nested gene-specific primers showed that rnCGM6 was present only in the testis, while rnCGM8 was present in the testis, intestine and lung. On the other hand rnCMG7 was found in all tissues examined. Furthermore, rnCGM7 transcript was present in all somatic cells examined whereas rnCGM6 was predominantly in myoid cells and rnCMG8 in Leydig cells. These results suggest that there is cell-specificity in the expression of PSGs in the rat testis and that the rat testis is a good model for studying the biological activities of the PSGs.

Amino Acid Sequence↗
PubMed1997

Effects of acute administration of dehydroepiandrosterone-sulfate on adipose tissue mass and cellularity in male rats.

OBJECTIVE: To determine if short term (2 week) treatment of growing male rats with low levels of dehydroepiandrosterone-sulfate (DHEAS) can reduce adiposity and serum triglycerides. DESIGN: Rats were administered either normal drinking water or drinking water supplemented with 10 (D10) or 100 (D100) micrograms/ml DHEAS for 14 d. SUBJECTS: Twenty-one male Sprague-Dawley rats (initial body weight 280 g). MEASUREMENTS: Adipocyte mass, size and number from three major fat depots (retroperitoneal, epididymal, inguinal); serum levels of triglycerides, insulin, IGF-1 and DHEAS; brown adipose tissue (BAT) mass, uncoupling protein content and enzyme activity; body weight gain, food and water consumption; carcass composition. RESULTS: DHEAS treatment had no effect on weight gain, food consumption or water intake. In contrast, rats treated with both levels of DHEAS had lighter fat pads, fewer epididymal and retroperitoneal adipocytes, less carcass lipid, lower levels of serum triglycerides and greater BAT mass and UCP content than control rats. Moreover, rats administered 100 micrograms/ml DHEAS had smaller and fewer epididymal adipocytes and fewer inguinal adipocytes than the D10 and the control rats. CONCLUSION: Acute treatment of growing male rats with low levels (10 micrograms/ml drinking water or 0.7 mg/kg body wt/d) of DHEAS reduces carcass lipid, adipose tissue mass and cellularity as well as serum triglycerides without altering food intake and body weight gain or causing hepatomegaly.

Adipocytes↗
PubMed1997

Response sensitivity and voltage gain of the rod- and cone-bipolar cell synapses in dark-adapted tiger salamander retina.

Response sensitivity and voltage gain of the rod- and cone-bipolar cell synapses in dark-adapted tiger salamander retina. J. Neurophysiol. 78: 2662-2673, 1997. Rods, cones, and bipolar cells were recorded in superfused, flat-mounted isolated retinas of the larval tiger salamander, Ambystoma tigrinum, under dark-adapted conditions. Voltage responses of 24 rods, 15 cones, and 41 bipolar cells in dark-adapted retinas to 500 nm light steps of various intensities were listed and fitted with hyperbolic functions, and their step sensitivities and relative sensitivities (log sigma) were estimated. In the linear response-intensity ranges, the step sensitivity of rods, SS(rod), is -1.0 mV photon-1 micron2 s or 0.034 mV Rh*-1 s rod and that of the cones, SS(cone), is approximately 0. 00146 mV photon-1 micron2 s or 0.000048 mV Rh*-1 s rod. The rod and cone responses were relatively homogenous with little variations in response amplitude and sensitivity. In contrast, bipolar cell responses were heterogenous with large variations in response amplitude and sensitivity. The maximum response amplitude of bipolar cells varied from 5 to 25 mV, and the relative response sensitivity (log sigma) varied >6 log units (-8.11 to -2.32). The step sensitivity of bipolar cells in the linear response-intensity range varied from 0.0000438 to 51.82 mV photon-1 micron2 s. Bipolar cells in dark-adapted tiger salamander retinas fell into two groups according to their relative sensitivities with very few cells falling in the intermediate light intensity region. The mixed bipolar cells (DBCM and HBCM) exhibited relative response sensitivity ranged from -8.11 to -5.54, and step sensitivity ranged from 1.22 to 51.82 mV photon-1 micron2 s. The cone-driven bipolar cells (DBCC and HBCC) exhibited relative response sensitivity ranged from -3.45 to -2.32, and step sensitivity ranged from 0.0000438 to 0. 00201 mV photon-1 micron2 sec. The chord voltage gain of the rod-DBCM or rod-HBCM synapses near the rod dark membrane potential ranged from 1.14 to 48.43 and that of the cone-DBCC or cone-HBCC synaptic gain near the cone dark membrane potential ranged from 0.03 to 1.38. The highest voltage gains were found near the rod or cone dark membrane potentials. By the use of linear subtraction method, we studied the synaptic inputs from cones to five mixed bipolar cells, and the voltage gains of the cone synapses in each of the bipolar cells were very close to the voltage gain of the rod synapses. This result suggests that although the responses of mixed bipolar cells are mediated mainly by rods when lights of short and medium wavelengths are used, their responses to long wavelength lights (>650 nm) are mediated by both rods and cones with comparable synaptic gains. Functional roles of the mixed and cone-driven bipolar cells in information processing in dark-adapted retinas are discussed.

Adaptation, Ocular↗
PubMed1997

Umbilical cord blood lead levels in Shanghai, China.

This study was designed to determine the cord blood lead (BPb) levels of babies born in one urban area of Shanghai, and to preliminarily identify the demographic, social environment and prenatal factors which have an effect on the cord BPb concentrations. From August to November 1993, umbilical cord blood samples were obtained from 605 live newborns in the Yangpu Maternal and Child Hospital. 257 samples were excluded from measurement because of clotting. In 348 cord samples, the geometric mean of cord BPb levels was 9.2 micrograms/dl, with a 95% confidence interval of the mean 8.86-9.54 (micrograms/dl). 142 babies (40.8%) had cord BPb levels of 10 micrograms/dl or greater. As a result of this high percentage of newborns with BPb levels equal to or greater than 10 micrograms/dl, we estimate that each year in the Shanghai City about 60,000 newborns are at risk for developing neuropsychological deficiencies caused by maternal lead exposure during pregnancy. To investigate the factors affecting cord blood levels, the subjects with levels greater than the 70th percentile (10.7 micrograms/dl) (n = 104) and less than the 30th percentile (7.4 micrograms/dl) (n = 104) were selected to compare the demographic, environment and prenatal medical history. Increased BPb levels at birth were associated with maternal passive smoking, a family member being occupationally exposed to lead, proximity to major traffic way, household coal combustion, neighborhood coal combustion, low level of maternal occupations, and the increasing occurrence of having the high lead foodstuff pidan (preserved duck egg) during pregnancy. We conclude that prenatal lead exposure has become an important health issue for young children in Shanghai.

Adult↗
PubMed1997

Characterization of the gamma-glutamyl carboxylase.

Studies on the vitamin K-dependent carboxylase are still in their infancy, but the cloning and purification of the enzyme have permitted a number of advances in our understanding of both the molecular biology, and mechanism of the carboxylase. Advances in our knowledge of the molecular biology of the carboxylase include chromosomal location, characterization of messenger RNA transcript(s) and the study of patients with carboxylase mutations. Our understanding of the mechanism of the carboxylase has been enhanced by the expression of peptides in E. coli which contain multiple carboxylation sites and more closely resemble the native carboxylase substrates. These peptides have been utilized to identify elements within the substrates which are critical for carboxylation and to demonstrate that the vitamin K-dependent carboxylase is one of the first examples of a processive post-translational modification enzyme.

Base Sequence↗
PubMed1996

A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism.

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Amino Acid Sequence↗